Alkali protease

ABSTRACT

Alkaline protease is produced by cultivating an alkaline protease-producing micro-organism from the genus Fusarium or Giberella in a proper growth supporting medium and the alkaline protease is recovered from the medium. The alkaline protease degrades protein under conditions of high pH and thus is useful in the formulation of detergent and other cleanser compositions.

llniled States Patent llsono et al.

[ 51- Mar. 28, 1972 ALKALI PROTEASE Inventors: Masao lsono, Nishinomiya, Hyogo; Katsumi Tomoda, Toyonaka; Koulclill Mlyata, Takatsuki; Kazutaka Maejima, Hyogo; Keisuke Tsubaki, Suita, Osaka, all of Japan Assignee: Takeda Chemical Industries, Ltd., Osaka,

Japan Filed: Apr. 30, 1969 Appl. No.: 820,485

Related U.S. Application Data Continuation-impart of Scr. No. 795,951, Feb. 3, 1969.

U.S. Cl. ..l95/62, 195/66 R llnt.Cl. ....C12d13/10 Field of Search 195/62, 66

References Cited UNITED STATES PATENTS 3,451,935 6/1969 Ro'ald et al l95/63 X OTHER PUBLICATIONS Narayanan et al., Chemical Abstracts Vol. 65, 1966 9252b to 9253a Primary Examiner-Lionel M. Shapiro Attorney-Wenderoth, Lind & Ponack ABSTRACT 2 Claims, 6 Drawing Figures PATENTEB-wee I972 8,652,399

' SHEET 1 BF 4 IQOF MASAO ISONO,

KATSUMI TOMODA,

KOUICHI MIYATA,

KAZUTAKA MAEJIMA AND KEISUKE TSUBAKI,

Inventors kbufiuun ll/v Attorneys PATENTEDMAR28 I972 SHEH 3 OF 4 Fig.3

Ill 33 (/o) kLIALLDV BALLV'IBII o oo menu T EMPERATURE (C) MASAO ISONO, KATSUMI TOMODA, KOUICHI MIYA'I'A, KAZUTAKA MAEJIMA & KEISUKE TSUBAKI,

Inventors XML PM Attorneys P'ATENTEU mm 28 1912 SHEET BF 4 TEMPERATURE (C F lg. 5

MASAO ISONO, KATSUMI TOMODA, KQUICHI MIYATA, KAZUTAKA MAEJIMA and KEISUKE TSUBAKI,

Inventors IOO Attorneys ALKALI PROTEASE The present application is a continuation-in-part of application Ser. No. 795,951, filed Feb. 3, I969.

The present invention relates to a novel alkali protease and to a process for producing the protease as well as to detergents and other cleansers containing the enzyme.

In the course of study for obtaining alkali proteases, it has been found that certain micro-organisms belonging to the genus F usan'um or the genus Giberella produce an alkali protease which exhibits a potent proteolytic activity around pH 11.

It has been found that the said protease actively degrades various kinds of protein under conditions of high pH even in the presence of surfactants and/or chelating agents, suggesting its applicability in the field of the detergent industry.

Heretofore, although certain enzymes have been applied to the laundry process, satisfactory results have not always been obtained. One of the possible causes therefor is that most cleansing solutions have a pH over pH 8. Another cause is that the enzymic activity is sometimes inhibited by surfactants and/or chelating agents contained in the detergents.

In this connection, further extensive studies have been carried out and a process established for producing a potent alkali protease on a commercial scale and manufactured detergents and other cleansers containing the enzyme. The enzyme is referred to herein as alkali protease".

The principal object of the present invention is therefore to provide an alkali protease which exhibits potent activity in the pH range from 8.0 to 12.0, particularly, from 10.0 to 11.5.

Another object is to provide detergents and other cleansers containing the alkali protease.

In order to realize the objects of the present invention, a strain of alkali protease-producing micro-organisms belonging to the genus Fusarium or the genus Gibberella is cultivated in a culture medium. Some typical micro-organisms producing the alkali protease are as follows:

(ATCC 20193 [PO numbers in the parentheses are the accession numbers at the Institute for Fermentation, Osaka, Japan; ATCC numbers are accession numbers at American Type Culture Collection, Rockville, Maryland.

Among these micro-organisms, Fusarium sp. S- 19-5 is one of the most useful strains for the production of the alkali protease. This strain was isolated from a soil sample by the inventors and shows the following microbiological characteristics:

1. Cultural characteristics (24C., 7 days) 1 Malt soup:

Good growth, white; flocculant pellicle formed; wrinkled on reverse; a scarlet-purple pigment in the medium.

2. Malt agar:

Good growth, white and flocculant; abundant aerial hyphae; wrinkled on reverse; a scarlet-purple pigment in the medium.

3. Czapecks solution:

Good growth, white; flocculant pellicle formed; wrinkled on reverse; substantially no pigment.

4. Czapecks agar:

Good growth, white and flocculant; wrinkled on reverse;

substantially no pigment.

5. Gelatin:

Good growth, white and flocculant; dark blackish purple pigment on reverse; weak liquefaction.

6. Potato dextrose:

Good growth, white and flocculant; the surface uneven,

raised; wrinkled on reverse, white to light purple; purple pigment liberated in the medium.

2. Microscopic characteristics:

On each of the above media, conidiophores develop from aerial hyphae, unbranched, l0 to 60 t long, 1.0 to 1.5 p. wide, hyaline. Conidia are produced apically on conidiophore and grouped in a slimy cluster, ovate or kidney-shaped,'oneto two-celled, rarely three-celled, 5 to 8 by l to 2 p, hyaline. No sexual state develops. Chlamydospore usually absent, sometimes present.

3. Physiological characteristics:

l. Condition for growth:

Hydrogen ion concentration: Preferably grows in alkaline media, optimal at between pH 8.0 to 9.0. Temperature; Optimal at about 24C..Allow to grow between 15 and 28C., although better growth is observed around at 15C. rather than at 28C. Oxygen requirement: Aerobic.

2. Gelatin: liquefaction:

Slight.

3. Utilization of ethyl alcohol:

Negative.

4. Degradation of pectin:

Very slight.

5. Degradation of tannin:

Negative.

6. Degradation of fats and oils:

Positive.

7. Utilization of carbohydrate:

Utilizes maltose, galactose, melibiose, sucrose, trehalose, fructose, mannose, rafiinose, dextrin, starch, glucose and lactose. Slightly utilizes inulin, xylose, arabinose and cellulose. According to Dictionary of the Fungi (G.C. Ainsworth, 5th edition), the said microbial characteristics indicate that this particular strain belongs to the genus Fusarium, the family Tuberculariaceae, the order Moniliales, the subclass Deutermycetes, the class Fungi Imperfecti.

Based on the Snyder & Hansen classification, Fusarium is divided into Section A, and A as follows:

A,: microconidia develop, usually one-celled.

A no microconidia develop or rarely produced, spindle,

comma or kidney-shaped, one to several-celled.

The organism, Fusarium sp. S-l9-5, produces microconidia dominantly and can be included in the Elegans Group of Section A. The Group Elegans is typified by F usarium oxysporum and includes 36 species which are all plant pathogens producing macroconidia in abundance.

The organism, F usarium sp. S-l9-5, however, produces only microconidia in most cases and the formation of macroconidia is very rare. This characteristic indicates the organism cannot be assigned to any of the hitherto known species of this group.

In order to cultivate an alkali protease-producing micro-organism, per se conventional culture media and conditions are used.

The medium may be used in either liquid or solid form. The culture may be effected under stationary conditions, however, it is more advantageous to adopt shake culture or aerobic culture methods.

In so far as an alkali protease-producing micro-organism may grow thereon, the medium may be of any type. For example, it may contain, as carbon sources, such materials as glucose, sucrose, dextrin, starch, cellulose, glycerol, sorbitol hydrocarbons and the like, and, as nitrogen sources, such materials as peptone, meat extract, yeast extract, dried yeast, soybean meal, soybean cake, rice bran, wheat bran, potato extract, casein, gluten, casein hydrolysate, corn steep liquor, urea, ammonium salts, nitrates and other organic or inorganic nitrogenous compounds. As inorganic salts, various phosphates, sulfates and hydrochlorides, for example, may be incorporated. Under certain circumstances, for the purpose of promoting growth of the micro-organism, various vitamins, amino acids, nucleic acids and their related compounds, etc., may be added. Depending upon the culture methods and conditions to be employed, the addition of a natural or synthetic defoaming agent, e.g., soybean oil or silicone oil, may be effective to increase accumulation of the enzyme.

In cultivating the micro-organisms, it is preferable to prepare a small scale preculture which is, in turn, inoculated into a main culture medium.

Culture conditions such as incubation temperature and time, pH of the medium, aeration rate, etc., should vary with the micro-organism and medium compositions to be used.

The requisite is that the conditions should be selected and controlled so that the accumulation of the alkali protease is maximal. In many instances the preferred conditions include the incubation temperature of from 20 to 30C, an incubation time of from 2 to 7 days, a medium pH of near 7, and an aeration rate of 0.5 to 1.5 liters per minute per liter of the medium.

Thus, the micro-organism accumulates a large amount of the alkali protease in the culture.

When a liquid medium is employed, the object enzyme occurs mostly in the liquid phase of the culture. Therefore, it is preferable to follow the steps of removing mycelia by filtration or centrifugation and, then, of recovering and isolating the enzyme from the filtrate or the supernatant fluid as the case may be.

When a solid medium is employed, it is usually preferable to subject the culture to extraction with water or an aqueous solution of an inorganic salt and, then, to recover the enzyme from the resulting extract.

In order to recover and isolate the enzyme from the culture filtrate, supernatant or extract, any of per se conventional isolation and purification means may be employed. These include salting out of enzyme with an inorganic salt such as sodium sulfate, ammonium sulfate or sodium chloride as well as fractional precipitation of enzyme by adding a suitable hydrophilic organic solvent such as methanol, ethanol or acetone. In addition, an enzyme solution may be concentrated under reduced pressure and/or demineralized by dialysis. It is also possible to employ means such as adsorption and desorption on calcium phosphate gel, alumina, bentonite, ion exchange resin, etc., chromatography using a cellulose derivative, e.g., diethylaminoethyl cellulose, gel-filtration, precipitation, electrodialysis, electrophoresis and removal of impurities as heavy metal complex.

The alkali protease produced and prepared in the foregoing manner is active over a broad pH range between 8.0 and 12.0, particularly in the pH region of 10.0 to l 1.5.

The optimal temperature for enzyme activity lies somewhere between about 20 and about 60C., particularly between about 40 and about 50C. This temperature range for the activity is in quite good accord with actual laundry conditions. Moreover, the activity of the alkali protease is not inhibited by surfactants and/or chelating agents which are principal ingredients in detergent products or cleansers.

In this invention, the alkali protease may be employed not only as a highly purified preparation but as a crude enzyme product and thus the product is obtained in a desired purity in accordance with the purpose; either pure or crude preparation of the enzyme may be used as an ingredient of detergent products or cleansers.

As surfactants contained in the detergent products, there may be mentioned for purpose of exemplification various compounds including anionic surfactants of the fatty acid salt type, sulfate type or sulfonate type, such as natural fatty acid soap(NS), alkylsulfate (DAE), olefine sulfate, n-a-olefine sulfonate (AOS), tetrapropylbenzene sulfonate (ABS) and n-alkylbenzene sulfonate (LAS); and nonionic surfactants of the ether type or ester type, such as polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyhydric alcohol alkyl esters, polyoxyethylene alkyl esters, sugar esters and the like.

The detergent products or cleansers may contain builders such as tri-polyphosphate, sulfates, carbonates, borates, as well as carboxymethyl cellulose, fluorescent dyes, scents, bleaching agents, e.g., perborates, chelating agents, e. g. N(CH COONa)3 skin-protective agents, e.g. dimethyllaurylaminoxide, disinfectants, e.g., tertiarily amine, and the like.

The alkali protease is mixed with other components of the product which may be a powdery, granular or liquid form and, when the resulting mixture is a liquid, it may be dried to a powdery or granular product by conventional means such as spray-drying.

Since there is no international system of unit for this kind of enzyme, it is difiicult to prescribe the portion of the enzyme in detergent products or cleansers in general.

For purposes of the present invention, activity of the alkali protease is assayed by the following modified Kunitz' method (J. Gen. Physiol. 30, 291, 1947). 1.0 ml. of a 2 percent auqeous solution of casein (Hammarstein's) and 0.5 ml. of 0.1 M glycine-NaOH buffer, pH 11.0, are mixed with 0.5 ml. of an enzyme solution. 2.0 ml. of the resulting mixture is allowed to incubate at 37C. for 20 minutes, and then the reaction is stopped by the addition of 3.0 ml. of a 5 percent aqueous solution of trichloracetic acid. The mixture is allowed to stand at 37C. for a further 30 minutes, whereby the undigested casein is thoroughly precipitated. The precipitated casein is filtered off and the resulting filtrate is subjected to optical density determination at 275 my, from which the amount of the digested casein is calculated as the amount of tyrosine. One unit of protease activity (PU) is defined as the amount of the enzyme which dissolved an amount of casein equivalent to 1.0 pg. of tyrosine per minute under the assay conditions. The specific activity of an enzyme sample is expressed as PU/mg.

The portion of the alkali protease to be incorporated in detergent products will vary with the type of product. In the case of a detergent to be used for washing cloth, the preferred amount will generally range from 5 to 10,000 in terms of units per gram of the detergent and for practical purposes from 10 to 5,000 units per gram.

An alkali protease-containing detergent prepared in the foregoing manner shows exceedingly powerful cleansing action against proteinous stains atributable to sweat, blood, broth and milk, which are hard to wash away with a conventional detergent.

Further, the detergent is employed in prewashing as well as in mainwashing.

In prewashing, soiled materials are usually soaked in a detergent solution at a room temperature for several hours, while the mainwashing is preferably carried out at an elevated temperature between 40 and 60C. for 1 hour or so.

Presently preferred embodiments of the invention are shown in the following examples, but they are not intended to be construed as a limitation on the present invention.

Throughout the specification, the abbreviations ml, mg", zg, z, m fl and LC" mean milliliter(s), milligram(s), microgram(s), micron(s), millimicron(s) and degree centigrade, respectively. Percentages are calculated on the weight per volume basis. In the following examples, parts by weight bear the same relation to parts by volume as do gram(s) to milliliter(s).

EXAMPLE I 500 parts by volume of a liquid medium, composed of 5 percent defatted soybean meal, 5 percent glucose, 2 percent sodium dihydrogen phosphate, and adjusted to pH 7, is dispensed in a fermenter (its capacity being 2,000 parts by volume), sterilized, inoculated with Fusarium sp. S-l9-5 (IFO 8884) (ATCC 20,192) and incubated at 28C for 5 days under aeration and agitation to prepare a seed culture.

The seed culture is inoculated to a fermenter (its capacity being 50,000 parts by volume) containing 30,000 parts by volume of the same liquid medium as above, and the fermenter is incubated at 25C. for 144 hours with the aeration rate of 45,000 parts by volume per minute under agitation of 500 rev./minute. During the incubation, foaming is suppressed by the addition of a suitable amount of soy bean oil from time to time.

5. Ultraviolet absorption spectrum (See FIG. 1):

Maximal absorption at 275-280 mp; Emm m" 7.0. 6. Infrared absorption spectrum (See FIG. 2):

Significant absorption bands in microns at 3.38, 6.05,

Changes of the pH value and the protease activity in the 6.55, 6.88,7.l5, 8.12, 9.30. course of the cultivation are shown as follows: 7. lsoelectric point (by paper electrophoresis):

Time of culture (hours) 0 18 30 42 64 66 78 90 122 144 pH 01 culture 7. 30 6. 40 6. 30 6. 12 6. 35 6. 72 7. 30 7. 09 7. 50 7. 50 Enzyme activity (PU/m 67 141 1, 430 2, 250 2,270 2, 520 2, 520 2,760 2, 540

EXAMPLE 2 About pH 1 1. 8. Solubilit The culture obtained after 144 hours as m Example IS Soluble ii. water and aqueous mineral salt solutions havcooled to about 5C. and, then, passed through a filter press in ionic Strength of 0 01 to o l Insoluble in with the filter aid, Hyflo Super-Cel (Johns-Manville Products mgthanol ethanol aceton and eth s Corp. U.S.A.), whereby the mycelia is removed. To the result- 9 PH activity z y ing 20,000 parts by volume of the filtrate is added 0.6-satu- Optimal PH at aboutll rated ammonium sulfate, and the salted-out precipitate is col- Temperature activity (See FIG lected by filtration with the filter aid. The resulting ammonium optimal temperature at about 6 sulfate precipitate containing the filter aid is dissolved in pH stability (See FIG. 5):

about 6,000 parts by volume of cold water and insoluble NO inactivation in the PH range from 5 to 10 percent materials are removed by filtration. 0.6-Saturated ammonium inactivation at PH 115 (Lhour incubation a sulfate is then added to the filtrate, so as to precipitate the en- 2 5 Temperature stability (See FIG 6):

Zyme again which in collected by centrifugation No inactivation below 55C.; 20 percent inactivation at solved in 1,000 parts by volume of cold water, dialyzed against and percent at 70C. (mqninute incubation at cold water by means of a fish-skin diaphragm for 4 days and PH 5).

lyophilized to give a crude enzyme powder. By the above procedure, 30 parts by weight of the crude enzyme powder 30 EXAMPLE4 with brownish color is obtained. The specific activity of this sample is about 980 PU/mg.

EXAMPLE 3 in 3,000 parts by volume of cold water is dissolved 30 parts by weight of the crude enzyme powder prepared in Example 2, and insoluble materials in the enzyme solution are filtered off with the filter aid to obtain a clear solution. 0.6-Saturated ammonium sulfate is added to the clear filtrate to give precipitate of the enzyme which is, then, redissolved in 1,500 parts by volume of cold water, decolorized with charcoal, dialyzed against cold water at 45C. for 4 days and liphilized to a powder. The above procedure yields 4.5 parts by weight of a partially purified enzyme powder with a specific activity of 1,580 PU/m g.

4.5 parts by weight of the enzyme powder, dissolved in 450 parts by volume of 0.01 M Tris-HCl buffer, pH 9.0, are dialyzed against 0.001 M Tris-l-lCl buffer, pH 9.0, for 3 days put on a column of diethylaminoethyl cellulose previously equilibrated with the Tris-HCl buffer and eluted with the same buffer. About 675 parts by volume of the enzyme rich fraction are collected, dialyzed against cold water for 3 days and lyophilized to a powder. The above procedure yields 0.35 part of purified enzyme in a powdery state with a specific activity of 5,400 PU/mg.

0.35 Part by weight of the purified enzyme powder, dissolved in 335 parts by volume of 0.01 M Tris-HCl buffer, pH 8.0, is chromatographed through a column of Sephadex G-100 previously washed with the Tris-HCl buffer. 62 parts by volume of the active fraction is collected, dialyzed against cold water for 3 days and lyophilized, whereupon 0.15 part by weight of a highly purified enzyme powder is obtained. The specific activity of this sample is 6,500 PU/mg.

The enzymological characteristics of this sample are as follows:

1. Color and shape: White powder. 2. A typical elementary analysis (percent):

C, 46.72; H, 6.59; N, 15.03. 3. Sedimentation constant (8 about 3.19 X 10. 4. Molecular weight (by Archibalds method):

2.65 X 10 (fl percent).

Influence of several surfactants on the alkali protease is examined as follows According to the formula given in Table l, five different detergent compositions are prepared where sodium salt of n-C, 18 natural fatty acid (NS), sodium n-C -alkylsulfate (DAS), sodium 'n-a-C olefinesulfonate (AOS), sodium tetrapropylbenzenesulfonate (ABS) and sodium n-C -alkylbenzenesulfonate are used as the surfactants, and these resulting detergent compositions are designated as NS-, DAS-, AOC ABS- and LAS-detergents, respectively.

TABLE 1 Component: Percent Surfactant Sodium tri-phosphate 40 Sodium sulfate 29 Sodium silicate 5 Carboxyrnethyl cellulose 1 5 mg. each of the respective detergents is dissolved in 2 ml. of an enzyme solution prepared by dissolving 2 mg. of the crude enzyme powder obtained in Example 2 into ml. of 0.05 M Tris-HCl buffer, pH 11.0, and the enzyme activity of the resulting solution is assayed according to the specified assay method. The results are shown in Table 2 which indicates that none of these detergents inhibits the enzyme activity.

Enzyme-containing detergent compositions are prepared by blending 0.05 g. of the crude enzyme powder obtained in Example 2 with 100 g. each of the above different detergents.

Relative activity: (PU/ml. with detergent) X100 a (P U/ml. without detergent) The cleaning activity of these enzyme-containing detergents are compared with those of the corresponding enzyme-free detergents under the laundry test conditions set forth in Table 3 TABLE 3 Soiled cloth (5 cm. x cm.).

The cleaning activity is evaluated by a panel monitation. Five pieces of the soiled cloth are assigned to the test of each detergent and, after the sequence of washing, rinsing and drying under the specified laundry test, the resulting washed cloths are scored by five judges according to the following standards of score.

Standards of score:

+2 Cleanliness of the cloth washed with an enzyme-containing detergent is definitely superior to that with the corresponding enzyme-free detergent.

+1 Cleanliness of the cloth washed with an enzyme-containing detergent is slightly superior to that with the corresponding enzyme-free detergent.

0 No significant difference is observed in cleanliness between the cloths.

The results of scoring are shown in Table 4, where the relative cleansing effects of the enzyme-containing detergents versus the corresponding enzyme-free detergents are presented, as to the respective types of detergent.

In the same manner as in Example 1, several micro-organisms belonging to the genus F usarium and the genus Gibberella are cultivated for 6 days.

The cultures are then centrifuged to give supernatant fluids which are used as enzyme solutions. To 2,000 parts by volume each of the enzyme solutions are added 5 parts by weight of the LAS-detergent described in Example 4, and protease activity of the resulting solution is determined by the specified assay method. The results are set forth in Table 5 indicating that the LAS-detergent does not inhibit any activity of the enzymes produced by those alkali protease-producing micro-organisms.

/ilzhrrz'lla saubimtti (IFO 6608) (ATCC 20193)" 523. 4

EXAMPLE 7 A liquid detergent for kitchen use: In 55 parts by volume of hot water at 60-65C. are dissolved 18 parts by weight of sodium tetrapropylbenzenesJfonate, 12 parts by weight of sodium n-C 2 allgilphenolethersulfate, 5 partsgy weight of lauryldrethanolami e and 10 parts by weight of 5 turn xylenesulfonate. After allowing to cool, the solution is supplemented with 0.5 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a liquid detergent composition for kitchen use.

EXAMPLE 8 Hair shampoo: In 64 parts by weight of hot water at 60-65 C. are dissolved 5 parts by weight of acetylated lanolin, 6 parts by weight of Alkylolamine (American Alcolac Corp.) and 25 parts by weight of Duponol XL (E. l. duPont de Nemours Company). After allowing to cool, the solution is supplemented with 0.2 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a hair shampoo.

EXAMPLE 9 1,000 parts by volume of a liquid medium, composed of 10 percent n-paraffine (C -C 5 percent defatted soybean meal, 0.05 percent MgSOfll-LO, 0.05 percent CaCl '2H O, 0.05 percent FeSO '7H,O, 0.8 percent K,HPO 0.3 percent KH PO 1.0 percent surface active agent and adjusted to pH 7, is dispensed in a fermenter (its capacity being 2,000 parts by volume), sterilized, inoculated with Fusarium sp. S-19-5 (ATCC 20,192) and incubated at 24C. for 6 days under aeration and agitation.

The filtrate of the culture broth shows 2,100 pu./ml. in protease activity.

The above obtained culture broth is subjected to the procedure as in Example 2 to yield 1.0 g. of a crude enzyme powder of 750 pu./mg. in a specific activity.

What is claimed is:

l. A method for producing alkali protease which comprises incubating an alkali protease-producing microorganism selected from the group consisting of Fusarium .rp. S-19-5 (ATCC 20,192), Fusarium oxysporum f. lini (IFO 5,880) and Gibberella saubinetti (ATCC 20,193) in a medium containing assimilable carbon sources and digestible nitrogen sources under aerobic conditions at an incubation temperature of about 20 C. to about 30 C. and at an incubation pH of from about 6 to about 9 until the alkali protease is substantially accumulated in the incubation medium, and recovering the accumulated alkali protease therefrom.

2. Alkali protease, produced by cultivating F usarium sp. S- 19-5 (ATCC 20,192), characterized by the following properties:

l. sedimentation constant (s of about 3.19 X 10 2. molecular weight of about 2.65 X 10 (by Archibalds method),

3. elementary analysis about 46.72 percent by weight carbon, about 6.59 percent by weight hydrogen and 15.3 percent by weight nitrogen,

4. maximum absorption, in its ultraviolet absorption spectrum at the wavelength of 275 to 280 mg, as indicated in FIG. 1,

5. infrared absorption spectrum as shown in FIG. 2, wherein the significant absorption bands in microns are as follows:

3.0 (strong), 3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium), 8.12 (medium), 9.30 (weak),

6. isoelectric point about pH 1 l,

7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and at a temperature from about 40 to about 50 C. as indicated in FIG. 4,

8. stable in the pH range from 5 to 8 in l-hour incubation at 9. 20 and percent loss of activity upon being heated at 65 and 70C., respectively, in 10-minute incubation at pH 5.0. 

2. Alkali protease, produced by cultivating Fusarium sp. S-19-5 (ATCC 20,192), characterized by the following properties:
 2. molecular weight of about 2.65 X 104 (by Archibald''s method),
 3. elementary analysis about 46.72 percent by weight carbon, about 6.59 percent by weight hydrogen and 15.3 percent by weight nitrogen,
 4. maximum absorption, in its ultraviolet absorption spectrum at the wavelength of 275 to 280 m Mu , as indicated in FIG. 1,
 5. infrared absorption spectrum as shown in FIG. 2, wherein the significant absorption bands in microns are as follows: 3.0 (strong), 3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium), 8.12 (medium), 9.30 (weak),
 6. isoelectric point about pH 11,
 7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and at a temperature from about 40* to about 50* C. as indicated in FIG. 4,
 8. stable in the pH range from 5 to 8 in 1-hour incubation at 37*C.,
 9. 20 and 80 percent loss of activity upon being heated at 65* and 70*C., respectively, in 10-minute incubation at pH 5.0. 